Proteolytic targeting chimera, methods of making and using the same

Abstract

The present application provides a kind of proteolysis targeting chimera and its preparation method and application, by containing phosphoramidite structure target protein ligand and containing phosphoramidite structure E3 ubiquitin ligase ligand is prepared by DNA automatic solid-phase synthesizer;Proteolysis targeting chimera is prepared by solid-phase synthesis method, with the advantages of fast, accurate, modular, automation, large batch preparation;By selecting suitable linker, increase the cell permeability of proteolysis targeting chimera, improve its ability to degrade target protein;In the hydroxyl modification of E3 ubiquitin ligase ligand, an enzyme activatable group, only when the corresponding enzyme removes the enzyme activatable group can release proteolysis targeting chimera to play its role, so as to carry out controllable degradation to target protein.The proteolysis targeting chimera prepared by the method provided by the present application can effectively inhibit the proliferation of human cervical cancer HeLa cells, has the advantages of strong specificity, good stability and high membrane permeability, and provides a new solution for tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of proteolysis targeting chimera, and particularly relates to a proteolysis targeting chimera, a preparation method and application thereof. BACKGROUND

[0002] Proteolysis targeting chimera (PROTAC) is a protein degradation technology that uses the ubiquitin proteasome system to degrade target proteins, thereby treating various diseases. Target protein ligands are combined with E3 ubiquitin ligase ligands through a linker to form a chimera that can recognize target proteins and induce E3 ubiquitin ligase to degrade target proteins. Unlike conventional small molecule inhibitors, which bind to the active site of a protein to affect the active function of the protein, PROTAC does not need to bind to the active site of the target protein, but only needs to form a ternary complex of the target protein ligand, the E3 ubiquitin ligase ligand, and the linker, which greatly expands the substrate range of the target protein and provides a new solution for the targeted degradation of undruggable proteins, bringing new breakthroughs in the treatment of related diseases. In the field of tumor treatment, many key signaling pathways and oncogenic proteins have become targets of proteolysis targeting chimeras, such as (MYC, p53, TAL1, STAT3). Compared with traditional drug methods, proteolysis targeting chimeras have higher targeting specificity and lower adverse reactions, and have great development potential.

[0003] However, there are still some challenges and problems in the development and application of proteolysis targeting chimeras. On the one hand, the preparation of proteolysis targeting chimeras relies on traditional chemical synthesis methods, which involve multiple chemical reactions and have the disadvantages of multiple steps, low efficiency, poor coupling precision, and low automation level. On the other hand, conventional proteolysis targeting chimeras have no selectivity between tumor cells and normal cells, resulting in significant toxic side effects, especially when they need to degrade specific proteins in specific cell types, their therapeutic potential is difficult to determine. These problems have brought certain difficulties to the research, optimization, and clinical application of proteolysis targeting chimera drugs.

[0004] Therefore, it is urgent to develop new synthesis methods and technologies to overcome these problems and promote the development of proteolysis targeting chimera technology. SUMMARY

[0005] In view of the prior art problems, the present application provides a proteolysis targeting chimera and a preparation method and application thereof, the proteolysis targeting chimera is prepared by synthesizing a target protein ligand containing a phosphoramidite structure and an E3 ubiquitin ligase ligand containing a phosphoramidite structure through a DNA solid-phase synthesizer; the proteolysis targeting chimera is prepared by a solid-phase synthesis method, and has the advantages of rapidness, precision, automation and large-batch preparation; by selecting a suitable linker, the cell permeability of the proteolysis targeting chimera is increased, and the ability of the proteolysis targeting chimera to degrade target proteins is improved; the hydroxyl group of the E3 ubiquitin ligase ligand can be modified with an enzyme-activatable group, and only when the corresponding enzyme removes the enzyme-activatable group can the proteolysis targeting chimera be released to play a role, so that the target protein is degraded. The proteolysis targeting chimera prepared by the method provided by the present application can effectively degrade target proteins in human cervical cancer HeLa cells and then inhibit the proliferation of the human cervical cancer HeLa cells, has the advantages of high specificity and high membrane permeability, and provides a new solution for tumor treatment.

[0006] In one aspect, the present application provides a preparation method of a proteolysis targeting chimera, which is prepared by a DNA automated solid-phase synthesis method, and comprises the following steps:

[0007] (1) preparing an E3 ubiquitin ligase ligand monomer containing a phosphoramidite structure and purifying the same;

[0008] (2) preparing a target protein ligand monomer containing a phosphoramidite structure and purifying the same;

[0009] (3) synthesizing the E3 ubiquitin ligase ligand monomer containing a phosphoramidite structure and the target protein ligand monomer containing a phosphoramidite structure on a solid-phase carrier through a DNA synthesizer, and after deprotection and purification treatment, a proteolysis targeting chimera is obtained.

[0010] The principle of DNA synthesis by a DNA solid-phase synthesizer is as follows: the terminal nucleotide of a DNA chain is fixed on a solid support (CPG) through a linker to form a nucleoside-derivatized solid support; nucleotides are sequentially added from the 3' end, each nucleotide forms a phosphite triester with the 5' end of the previous nucleotide, and then is oxidized into a phosphate triester; after each round of synthesis, the protecting group DMT on the 5' end hydroxyl group is removed, and the next round of synthesis is continued. The DNA synthesized by the above solid-phase synthesis method has a raw material nucleotide containing a phosphoramidite structure, so the present application designs an E3 ubiquitin ligase ligand containing a phosphoramidite structure by referring to this principle. The ligand molecule is first connected to a universal solid support, and then subjected to oxidation, capping, deprotection and other treatments, and then reacts with a target protein ligand containing a phosphoramidite structure to form a phosphate ester structure. All reactions are completed by automatic modularization on a DNA synthesizer. The phosphate ester includes a phosphodiester, a phosphorothioate, and a phosphotriester, which couples the E3 ubiquitin ligase ligand and the target protein ligand together to obtain a proteolysis targeting chimera, and finally the proteolysis targeting chimera is cut off from the CPG.

[0011] Therefore, the E3 ubiquitin ligase ligand and the target protein ligand prepared in advance must have a phosphoramidite structure, so as to synthesize a proteolysis targeting chimera by a DNA solid-phase synthesizer through a solid-phase synthesis method, and then purify the proteolysis targeting chimera by high performance liquid chromatography (HPLC) after deprotection. If the target protein ligand prepared in advance does not contain a phosphoramidite monomer structure, the proteolysis targeting chimera cannot be prepared by this method. Compared with the method of chemically synthesizing a proteolysis targeting chimera, the solid-phase synthesis method of synthesizing a proteolysis targeting chimera avoids the limitations of multiple steps, long time consumption, high cost and inaccuracy, and has the advantages of rapidity, accuracy, modularity, automation and large-scale preparation.

[0012] Further, the purification method of steps (1) and (2) is purification by a chromatography column, and the eluent of the chromatography column contains triethylamine.

[0013] In some modes, the chromatography column is a silica gel chromatography column, and 1% triethylamine needs to be added to the eluent during purification. If 1% triethylamine is not added to the eluent, the yield of the E3 ubiquitin ligase ligand and the target protein ligand will be reduced by 20%-30%; or 1% triethylamine is replaced by other substances, such as 1% ammonia, which will also cause the yield of the product to decrease.

[0014] Further, the solid support of step (3) includes a universal solid support or a solid support containing an enzyme-activatable group, and the enzyme-activatable group includes one or more of a phosphate group and a nitro group.

[0015] In the process of synthesizing proteolytic targeting chimera by DNA solid-phase synthesizer, the proteolytic targeting chimera obtained by adding a general solid-phase carrier does not contain an enzyme-activatable group, and it can play a role as soon as it enters tumor cells; while the proteolytic targeting chimera obtained by adding a solid-phase carrier containing an enzyme-activatable group contains an enzyme-activatable group, which is modified on the hydroxyl group of the E3 ubiquitin ligase ligand, and the proteolytic targeting chimera needs to be removed from the enzyme-activatable group by the corresponding enzyme before it can play a role in tumor cells. Compared with the proteolytic targeting chimera not modified by the enzyme-activatable group, the former not only has selectivity for tumor cells and can only play a role in tumor cells that can remove the enzyme-activatable group, but also has selectivity for degrading proteins and can only degrade proteins combined with the targeting protein ligand.

[0016] In another aspect, a proteolytic targeting chimera is prepared by a solid-phase synthesis method, and the proteolytic targeting chimera has the following structural formula:

[0017]

[0018] Wherein, L is a linker, the left side of L is a targeting protein ligand, the right side of L is an E3 ubiquitin ligase ligand, and R is a substituent.

[0019] In theory, the related proteins in the pathogenesis of diseases can be designed according to their structures to design corresponding targeting protein ligands, and the targeting protein ligands and E3 ubiquitin ligase ligands can be coupled by a solid-phase synthesis method to prepare an enzyme-activatable proteolytic targeting chimera.

[0020] The linker plays a key role in the entry of the proteolytic targeting chimera into cells, and the charge and structure of the linker affect the ability of the proteolytic targeting chimera to enter cells and its stability in the body environment. The present application solves the problem of poor membrane permeability of the proteolytic targeting chimera by screening the linker.

[0021] The targeting protein ligand provided by the present application targets BRD4 protein, and BRD4 protein is bromodomain protein 4, which plays a key role in the process of gene transcription and epigenetic regulation. Its N-terminal can recognize and selectively bind to specific acetylated lysine residues at the tail of the protein, thereby regulating the binding of chromatin remodeling factors, transcription factors and other related proteins to specific gene transcription sites, and plays an important role in the infiltration, metastasis and malignant development of tumor cells. The occurrence and development of various malignant tumors such as cervical cancer, osteosarcoma, lung cancer, breast cancer, hematological tumors and liver cancer are related to BRD4 dysfunction. Therefore, BRD4 protein is an ideal drug target.

[0022] Further, the linker comprises one or more of a phosphodiester, a phosphorothioate, a phosphotriester.

[0023] Further, the linker is a phosphotriester.

[0024] Further, the substituent comprises one or more of a hydroxyl group, a phosphate group, a nitro group.

[0025] In some aspects, the substituent is preferably a phosphate group.

[0026] Further, the proteolysis targeting chimera comprises any one or more of the following A, B, C, D formulae:

[0027]

[0028]

[0029] The linker of the proteolysis targeting chimera A is a phosphodiester, the linker of the proteolysis targeting chimera B is a phosphorothioate, and the linkers of the proteolysis targeting chimera C and the proteolysis targeting chimera D are phosphotriesters.

[0030] Further, the proteolysis targeting chimera comprises any one or more of the C, D formulae.

[0031] In another aspect, use of a proteolysis targeting chimera for the manufacture of an anti-tumor medicament, the proteolysis targeting chimera comprising a targeting protein ligand, a linker comprising one or more of a phosphodiester, a phosphorothioate, a phosphotriester, and an E3 ubiquitin ligase ligand that can modify an enzyme activatable group.

[0032] In some aspects, the ability of the proteolysis targeting chimeras A, B, C containing different linkers and a commercially available proteolysis targeting chimera MZ1 to inhibit the growth of human cervical cancer HeLa cells and degrade BRD4 protein in human cervical cancer HeLa cells is detected by experiments, which proves that the proteolysis targeting chimera A containing a phosphodiester linker and the proteolysis targeting chimera B containing a phosphorothioate linker have poor cell permeability and poor inhibitory effect on human cervical cancer HeLa cells, while the proteolysis targeting chimera C containing a phosphotriester linker and MZ1 containing a polyethylene glycol linker have relatively good inhibitory effect on human cervical cancer HeLa cells. The proteolysis targeting chimera C containing a phosphotriester linker has stronger inhibitory effect on human cervical cancer HeLa cells and stronger ability to degrade BRD4 protein in human cervical cancer HeLa cells than MZ1, and the proteolysis targeting chimera C has less inhibitory effect on other tumor cells than human cervical cancer HeLa cells.

[0033] In some modes, the ability of the alkaline phosphatase-activatable proteolysis targeting chimera D and the unmodified enzyme-activatable group proteolysis targeting chimera C to inhibit the proliferation of human osteosarcoma cell Saos-2 cells and human osteosarcoma cell U-2OS cells, respectively, is detected by experiments, which proves that the alkaline phosphatase-activatable proteolysis targeting chimera D can only efficiently inhibit the proliferation of Saos-2 cells with high alkaline phosphatase expression, and the unmodified enzyme-activatable group proteolysis targeting chimera C has good inhibitory effect on the proliferation of Saos-2 cells with high alkaline phosphatase expression and U-2OS cells with low alkaline phosphatase expression, indicating that the alkaline phosphatase-activatable proteolysis targeting chimera D can specifically inhibit the proliferation of tumor cells with high alkaline phosphatase expression.

[0034] In some modes, the ability of the alkaline phosphatase-activatable proteolysis targeting chimera D and the unmodified enzyme-activatable group proteolysis targeting chimera C to degrade BRD4 protein in human osteosarcoma cell Saos-2 cells and human osteosarcoma cell U-2OS cells, respectively, is detected by experiments, which proves that the alkaline phosphatase-activatable proteolysis targeting chimera D only has high-efficiency degradation ability of BRD4 protein in Saos-2 cells with high alkaline phosphatase expression, and the unmodified enzyme-activatable group proteolysis targeting chimera C has high-efficiency degradation ability of BRD4 protein in Saos-2 cells with high alkaline phosphatase expression and U-2OS cells with low alkaline phosphatase expression, indicating that the alkaline phosphatase-activatable proteolysis targeting chimera D has specific selection for tumor cells and can efficiently degrade BRD4 protein in specific cells.

[0035] The present application has the following beneficial effects:

[0036] 1. The present application provides a proteolysis targeting chimera, which is synthesized by a DNA solid-phase synthesizer with a target protein ligand containing a phosphoramidite structure and an E3 ubiquitin ligase ligand containing a phosphoramidite structure, and the proteolysis targeting chimera is prepared by a solid-phase synthesis method, which has the advantages of rapidness, precision, automation and large-scale preparation.

[0037] 2. The cell permeability of the proteolysis targeting chimera is increased by screening a linker, and the ability of the proteolysis targeting chimera to degrade target proteins is improved.

[0038] 3. The hydroxyl group of the E3 ubiquitin ligase ligand can be modified with an enzyme-activatable group, and only when the corresponding enzyme in the cell removes the enzyme-activatable group can the proteolysis targeting chimera be released to play its role, thereby degrading the target protein.

[0039] 4. The protein hydrolysis targeting chimera C prepared by the method has the advantages of high specificity, good stability and high membrane permeability, and can effectively degrade the target protein in human cervical cancer HeLa cells and inhibit the proliferation of the target protein, thereby providing a new solution for tumor treatment.

[0040] 5. The protein hydrolysis targeting chimera D prepared by the method can specifically inhibit the proliferation of tumor cells with high expression of alkaline phosphatase. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The hydrogen spectrum of compound 2 in Example 1 is shown in the figure;

[0042] Figure 2 The carbon spectrum of compound 2 in Example 1 is shown in the figure;

[0043] Figure 3 The hydrogen spectrum of compound 3 in Example 1 is shown in the figure;

[0044] Figure 4 The carbon spectrum of compound 3 in Example 1 is shown in the figure;

[0045] Figure 5 The hydrogen spectrum of compound 4 in Example 1 is shown in the figure;

[0046] Figure 6 The carbon spectrum of compound 4 in Example 1 is shown in the figure;

[0047] Figure 7 The phosphorus spectrum of compound 4 in Example 1 is shown in the figure;

[0048] Figure 8 The hydrogen spectrum of compound 6 in Example 1 is shown in the figure;

[0049] Figure 9 The carbon spectrum of compound 6 in Example 1 is shown in the figure;

[0050] Figure 10 The hydrogen spectrum of compound 7 in Example 1 is shown in the figure;

[0051] Figure 11 The carbon spectrum of compound 7 in Example 1 is shown in the figure;

[0052] Figure 12 The hydrogen spectrum of compound 8 in Example 1 is shown in the figure;

[0053] Figure 13 The carbon spectrum of compound 8 in Example 1 is shown in the figure;

[0054] Figure 14 The phosphorus spectrum of compound 8 in Example 1 is shown in the figure;

[0055] Figure 15 The hydrogen spectrum of compound 10 in Example 1 is shown in the figure;

[0056] Figure 16 Carbon spectrum of compound 10 in Example 1;

[0057] Figure 17 Phosphorus spectrum of compound 10 in Example 1;

[0058] Figure 18 Figure showing the results of the HeLa cell proliferation inhibition experiment of proteolysis targeting chimera A, B, C and MZ1 in Example 2;

[0059] Figure 19 Liquid chromatogram for the hydrophobicity comparison of proteolysis targeting chimera MZ1 and C in Example 2;

[0060] Figure 20 WB experiment graph showing the degradation of BRD4 protein in HeLa cells by proteolysis targeting chimera MZ1 and C in Example 3;

[0061] Figure 21 Curve graph showing the degradation of BRD4 protein in HeLa cells by proteolysis targeting chimera MZ1 in Example 3;

[0062] Figure 22 Curve graph showing the degradation of BRD4 protein in HeLa cells by proteolysis targeting chimera C in Example 3;

[0063] Figure 23 Mass spectrum of proteolysis targeting chimera C in Example 4;

[0064] Figure 24 Mass spectrum of proteolysis targeting chimera D in Example 4;

[0065] Figure 25 Mass spectrum of proteolysis targeting chimera C obtained by dephosphorylation of proteolysis targeting chimera D in Example 4;

[0066] Figure 26 Liquid chromatogram obtained after HPLC treatment for verifying whether alkaline phosphatase can dephosphorylate proteolysis targeting chimera D to obtain proteolysis targeting chimera C in Example 4;

[0067] Figure 27 Liquid chromatogram obtained after HPLC treatment for verifying whether alkaline phosphatase of different concentrations can dephosphorylate proteolysis targeting chimera D to obtain proteolysis targeting chimera C in Example 4;

[0068] Figure 28 Liquid chromatogram obtained after HPLC treatment for verifying whether alkaline phosphatase inhibitor Na3VO4 can inhibit the dephosphorylation of D to obtain C in Example 4;

[0069] Figure 29 Figure 6 is a graph showing the quantification of extracted C by HPLC chromatogram peak area determination for Example 5;

[0070] Figure 30 Figure 7 is a graph showing the stability profile of extracted C by HPLC chromatogram peak area determination for Example 5;

[0071] Figure 31 Figure 8 is a graph showing the quantification of extracted BRD4 protein degrader (MZ1) by HPLC chromatogram peak area determination for Example 5;

[0072] Figure 32 Figure 9 is a graph showing the stability profile of extracted MZ1 by HPLC chromatogram peak area determination for Example 5;

[0073] Figure 33 Figure 10 is a graph showing the experimental results of alkaline phosphatase expression testing in Saos-2 cells and U-2OS cells for Example 7;

[0074] Figure 34 Figure 11 is a graph showing the experimental results of proteolysis targeting chimera C and D on Saos-2 cell proliferation inhibition for Example 7;

[0075] Figure 35 Figure 12 is a graph showing the experimental results of proteolysis targeting chimera C and D on U-2OS cell proliferation inhibition for Example 7;

[0076] Figure 36 Figure 13 is a graph showing the WB experimental results of proteolysis targeting chimera C and D on BRD4 protein degradation in Saos-2 cells for Example 7;

[0077] Figure 37 Figure 14 is a graph showing the curve of proteolysis targeting chimera C on BRD4 protein degradation in Saos-2 cells for Example 7;

[0078] Figure 38 Figure 15 is a graph showing the curve of proteolysis targeting chimera D on BRD4 protein degradation in Saos-2 cells for Example 7;

[0079] Figure 39 Figure 16 is a graph showing the WB experimental results of proteolysis targeting chimera C and D on BRD4 protein degradation in U-2OS cells for Example 7;

[0080] Figure 40 Figure 17 is a graph showing the curve of proteolysis targeting chimera C on BRD4 protein degradation in U-2OS cells for Example 7;

[0081] Figure 41Graph of BRD4 protein degradation in U-2OS cells by proteolysis targeting chimera D of Example 7. DETAILED DESCRIPTION

[0082] The application will be further described in conjunction with the drawings and examples, it is pointed out that the following examples are intended to facilitate an understanding of the application and are not intended to limit it in any way.

[0083] Example 1, Preparation of proteolysis targeting chimera

[0084] 1, The preparation method of proteolysis targeting chimera is as follows:

[0085] 1) Synthesis of E3 ubiquitin ligase ligand containing phosphoramidite structure

[0086] The synthesis route reaction formula is as follows:

[0087]

[0088] The specific steps are as follows:

[0089] Synthesis of compound 2: under nitrogen protection, compound 1 (1 g, 2.08 mmol), 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1.19 g, 3.12 mmol) and 6-hydroxyhexanoic acid (0.303 g, 2.29 mmol) were dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), and the reaction was stirred overnight. After the reaction was completed, it was extracted twice with dichloromethane and saturated sodium bicarbonate solution, and the organic layer was collected. Anhydrous sodium sulfate was added for drying, and after rotary evaporation, it was purified by a chromatographic column to obtain white foamy solid compound 2 (0.761 g, 1.36 mmol, 65.38%). 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.39 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H), 5.11 (d, J = 3.5 Hz, 1H), 4.96 - 4.86 (m, 1H), 4.51 (d, J = 9.3 Hz, 1H), 4.42 (t, J = 8.1 Hz, 1H), 4.34 (t, J = 5.1 Hz, 1H), 4.30 - 4.25 (m, 1H), 3.60 (d, J = 3.4 Hz, 2H), 3.39 - 3.35 (m, 2H), 2.45 (s, 3H), 2.23 (dd, J = 14.6, 6.7 Hz, 1H), 2.12 (d, J = 7.9 Hz, 1H), 2.01 (ddd, J = 10.2, 7.7, 2.3 Hz, 1H), 1.78 (s, 1H), 1.56 - 1.44 (m, 2H), 1.37 (d, J = 7.1 Hz, 3H), 1.27 (d, J = 7.7 Hz, 2H), 1.25 (s, 2H), 0.93 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 172.06, 170.65, 169.61, 151.52, 147.77, 144.70, 131.13, 129.69, 128.84, 126.39, 68.77, 60.64, 58.55, 56.34, 47.70, 37.75, 35.21, 34.99, 32.30, 26.46, 25.25, 22.48, 16.01. The hydrogen spectrum of compound 2 is shown in Figure 1 The carbon spectrum is shown in Figure 2

[0090] Synthesis of compound 3: Compound 2 (0.761 g, 1.36 mmol) was dissolved in 10 mL of anhydrous pyridine under nitrogen protection, and 4,4'-dimethoxytrityl chloride (DMT-C1) (0.553 g, 1.632 mmol) was added, and the reaction was stirred overnight. After the reaction was completed, the pyridine was first removed by rotary evaporation, and after drying, the compound 3 (0.899 g, 1.04 mmol, 76.5%) was obtained as a white foamy solid after purification by column chromatography. 1 ​H NMR (400 MHz, Chloroform-d) δ 8.67 (s, 1H), 7.46 - 7.39 (m, 5H), 7.36 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 2.1 Hz, 2H), 7.31 - 7.28 (m, 2H), 7.20 (d, J = 7.2 Hz, 1H), 6.90 - 6.71 (m, 4H), 6.16 (d, J = 8.7 Hz, 1H), 5.20 - 5.01 (m, 1H), 4.69 (t, J = 7.9 Hz, 1H), 4.56 (d, J = 8.8 Hz, 1H), 4.49 (s, 1H), 4.08 (d, J = 11.5 Hz, 1H), 3.78 (s, 6H), 3.59 (dd, J = 11.3, 3.7 Hz, 1H), 3.03 (t, J = 6.5 Hz, 2H), 2.94 (d, J = 7.3 Hz, 1H), 2.52 (s, 3H), 2.18 (t, J = 7.6 Hz, 2H), 2.07 - 1.99 (m, 1H), 1.59 (q, J = 6.8, 6.4 Hz, 4H), 1.47 (d, J = 6.9 Hz, 3H), 1.30 (t, J = 7.3 Hz, 2H), 1.03 (s, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 173.82, 172.17, 169.78, 158.39, 150.45, 148.60, 145.44, 143.23, 136.74, 130.11, 129.69, 128.28, 127.82, 126.70, 126.57, 113.09, 85.80, 70.05, 63.27, 58.56, 57.55, 56.76, 55.31, 36.60, 35.49, 35.16, 29.93, 26.63, 26.17, 25.66, 22.35, 16.21. The hydrogen spectrum of compound 3 is shown in Figure 3 and the carbon spectrum is shown in Figure 4 .

[0091] Synthesis of compound 4: Compound 3 (0.899 g, 1.04 mmol) was dissolved in 15 mL of dry dichloromethane under anhydrous and oxygen free environment and under nitrogen protection, N,N-diisopropylcarboxamide (DIPEA) (0.403 g, 3.12 mmol) was added dropwise. After cooling in an ice bath for five minutes, 2-cyanoethyl-N,N diisopropyl chlorophosphoramidite (0.369 g, 1.56 mmol) was added, the reaction was fast and needed to be monitored in real time using thin layer chromatography (TLC). After the reaction was complete, extraction was performed using dichloromethane and saturated potassium chloride solution, the organic layer was collected, dried by adding anhydrous sodium sulfate, spun down quickly, dry loaded, purified using silica gel column, packed and eluted using eluent prepared by mixing 1 : 1 by volume of petroleum ether and ethyl acetate, and adding 1% triethylamine. The product was spun down using a fast speed, and after spinning, it was dried using a vacuum pump and preserved using nitrogen to prevent oxidation. Compound 4 (0.828 g, 0.78 mmol, 75%) was obtained as a white foamy solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.67 (s, 1H), 7.45 - 7.39 (m, 4H), 7.38 - 7.28 (m, 7H), 7.27 (s, 1H), 7.20 (d, J = 7.2 Hz, 1H), 6.81 (d, J = 8.9 Hz, 4H), 5.16 - 5.01 (m, 1H), 4.77 - 4.55 (m, 3H), 3.92 - 3.81 (m, 1H), 3.78 (s, 6H), 3.76 - 3.64 (m, 2H), 3.54 (ddd, J = 14.4, 10.3, 7.0 Hz, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.62 (t, J = 6.1 Hz, 2H), 2.53 (s, 3H), 2.18 (q, J = 7.7 Hz, 2H), 1.75 (s, 2H), 1.61 (td, J = 11.9, 9.7, 4.8 Hz, 4H), 1.47 (d, J = 6.9 Hz, 3H), 1.40 (d, J = 4.0 Hz, 2H), 1.18 - 1.09 (m, 12H), 1.01 (s, 9H). 13C NMR (101 MHz, Chloroform-d) δ 172.19, 171.94, 169.52, 158.30, 150.30, 148.51, 145.36, 143.19, 143.05, 136.69, 130.01, 129.59, 128.19, 127.72, 126.60, 126.46, 118.15, 117.80, 112.98, 85.69, 71.50, 63.21, 58.79, 58.08, 56.73, 55.21, 53.46, 48.89, 36.29, 35.07, 29.91, 26.49, 26.22, 25.80, 24.57, 22.25, 20.29, 16.13. 31 H NMR (400 MHz, Chloroform-d) δ 7.43 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 4.60 (t, J = 6.9 Hz, 1H), 3.71 (dd, J = 16.8, 7.1 Hz, 1H), 3.59 (dd, J = 16.8, 6.7 Hz, 1H), 2.68 (s, 3H), 2.41 (s, 3H), 1.69 (s, 3H). Figure 5 Figure 6 Figure 7

[0092] 2) Synthesis of target protein ligand containing phosphoramidite structure

[0093] There are two synthesis path reaction formulas, which are as follows:

[0094]

[0095] The specific steps are as follows:

[0096] Synthesis of compound 6: under nitrogen protection, compound 5 (2 g, 4.38 mmol) was dissolved in 24 mL of dichloromethane, 6 mL of trifluoroacetic acid (8.778 g, 77.19 mmol) was added dropwise, and the reaction was stirred at room temperature for 6 h. After spinning dry, purification by column chromatography gave yellow foam solid compound 6 (1.646 g, 4.11 mmol, 94.1%). 1 H NMR (400 MHz, Chloroform-d) δ 7.43 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 4.60 (t, J = 6.9 Hz, 1H), 3.71 (dd, J = 16.8, 7.1 Hz, 1H), 3.59 (dd, J = 16.8, 6.7 Hz, 1H), 2.68 (s, 3H), 2.41 (s, 3H), 1.69 (s, 3H). 13 ​​​C NMR (101 MHz, Chloroform-d) δ 173.22, 164.15, 155.40, 149.99, 136.95, 136.36, 131.93, 131.21, 131.01, 130.54, 129.94, 128.76, 45.72, 14.43, 13.14, 11.74. The hydrogen spectrum of compound 6 is shown in Figure Figure 8 The carbon spectrum is shown in Figure Figure 9 .

[0097] Synthesis of compound 7: Compound 6 (1.646 g, 4.11 mmol) and 2-(7-oxidebenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (2.344 g, 6.165 mmol) were dissolved in 20 mL of dry N,N-dimethylformamide (DMF) under nitrogen protection, 3-amino-1-propanol (0.37 g, 4.932 mmol) and N,N-diisopropylformamide (DIPEA) (1.328 g, 10.275 mmol) were added dropwise, and the reaction was stirred overnight. After the reaction was completed, it was extracted with ethyl acetate and saturated sodium chloride solution, the organic layer was collected, dried by adding anhydrous sodium sulfate, and after rotary evaporation, it was purified by column chromatography to obtain compound 7 (1.616 g, 3.53 mmol, 85.89%) as a white foamy solid. 1 H NMR (400 MHz, Chloroform-d) δ 7.40 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 4.66 (dd, J = 7.8, 6.4 Hz, 1H), 3.63 (d, J = 6.8 Hz, 2H), 3.56 (dd, J = 14.5, 7.8 Hz, 1H), 3.46 (q, J = 6.1 Hz, 2H), 3.40 (dd, J = 14.5, 6.4 Hz, 1H), 2.67 (s, 3H), 2.40 (s, 3H), 1.75 - 1.69 (m, 2H), 1.67 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 171.36, 164.08, 155.68, 149.99, 136.84, 136.48, 131.88, 131.00, 130.65, 129.85, 128.72, 58.82, 54.28, 45.57, 39.00, 32.09, 14.41, 13.12, 11.81. The hydrogen spectrum of compound 7 is shown in Figure Figure 10 The carbon spectrum is shown in Figure Figure 11 .

[0098] Synthesis of compound 8: Compound 7 (0.762 g, 1.66 mmol) was dissolved in 15 mL of dry dichloromethane in anhydrous and oxygen free environment under nitrogen atmosphere, N,N-diisopropylformamide (DIPEA) (0.644 g, 4.98 mmol) was added drop wise, after cooling in ice bath for five minutes 2-cyanoethyl-N,N diisopropyl chlorophosphoramidite (0.589 g, 2.49 mmol) was added, the reaction was fast and TLC was used to monitor the progress of the reaction in real time. After completion of the reaction, extraction was done with dichloromethane and saturated potassium chloride solution, the organic layer was collected, dried by adding anhydrous sodium sulfate and was spun down, dried loaded on silica gel column and purified. The column was packed and eluted with eluent prepared by mixing petroleum ether and ethyl acetate in the ratio of 1 : 1 and adding 1% triethylamine. The product was spun down at a fast speed, after spinning the product was dried by using a vacuum pump and was preserved under nitrogen atmosphere to prevent oxidation of the product. The product compound 8 (0.5 g, 0.759 mmol, 45%) was obtained as a white foamy solid. 1 HNMR (400 MHz, Chloroform-d) δ 7.42 - 7.36 (m, 2H), 7.32 (dd, J = 8.8, 1.1 Hz, 2H), 4.63 (t, J = 7.0 Hz, 1H), 3.96 - 3.77 (m, 2H), 3.77 - 3.66 (m, 2H), 3.65 - 3.55 (m, 2H), 3.54 - 3.41 (m, 2H), 3.35 (dddd, J = 17.8, 14.3, 6.6, 3.8 Hz, 2H), 2.68 (d, J = 6.4 Hz, 1H), 2.65 (s, 3H), 2.62 (d, J = 6.4 Hz, 1H), 2.39 (s, 3H), 1.91 - 1.79 (m, 2H), 1.66 (s, 3H), 1.17 (dd, J = 6.7, 3.8 Hz, 12H). 13 C NMR (101 MHz, Chloroform-d) δ 170.45, 163.92, 155.67, 149.93, 136.77, 136.64, 132.13, 130.93, 130.83, 130.48, 129.85, 128.73, 117.76, 61.57, 58.24, 54.43, 43.13, 37.12, 30.68, 24.70, 20.41, 14.42, 13.11, 11.86. 31 P NMR (162 MHz, Chloroform-d) δ 147.76. The hydrogen spectrum of compound 8 is shown in Figure 12 , the carbon spectrum is shown in Figure 13 , and the phosphorus spectrum is shown in Figure 14 .

[0099] Synthesis of compound 9: Compound 7 (0.74 g, 1.62 mmol) was dissolved in 24 mL of dry dichloromethane under anhydrous and oxygen free conditions and under nitrogen protection, N,N-diisopropylformamide (DIPEA) (0.229 g, 1.77 mmol) was added dropwise and cooled in a ice-salt bath for ten minutes. Bis(diisopropylamino)chlorophosphonium (0.472 g, 1.77 mmol) was dissolved in 10 mL of dry dichloromethane and the solution of bis(diisopropylamino)chlorophosphonium in dry dichloromethane was added dropwise slowly, the reaction was fast and TLC was used to monitor the reaction progress in real time, after the reaction was complete the crude product compound 9 was used directly for the next reaction.

[0100] Synthesis of compound 10: To the crude compound 9, dry isobutanol (0.131 g, 1.77 mmol) and 5-(ethylthio)-lH-tetrazole (0.23 g, 0.81 mmol, 0.25 M prepared in advance by dissolving in dry acetonitrile, and degassed) were added under anhydrous and oxygen free conditions and under nitrogen protection, TLC was used to monitor the reaction progress in real time, after the reaction was complete the product was extracted with dichloromethane and saturated potassium chloride solution, the organic layer was collected, dried with anhydrous sodium sulfate, spun down quickly, and loaded on silica gel column for purification. The column was packed and eluted with an eluent prepared by adding 1% triethylamine to ethyl acetate. The product was spun down quickly, and after spinning down the product was dried with a vacuum pump and stored under nitrogen to prevent oxidation. The product compound 10 was obtained as a white foamy solid (0.426 g, 0.644 mmol, 39.8%). 1 H NMR (400 MHz, Chloroform-d) δ 7.38 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 4.63 (t, J = 6.9 Hz, 1H), 3.78 - 3.61 (m, 2H), 3.62 - 3.53 (m, 2H), 3.48 (ddd, J = 19.5, 10.5, 4.2 Hz, 2H), 3.44 - 3.36 (m, 2H), 3.36 - 3.28 (m, 2H), 2.64 (s, 3H), 2.38 (s, 3H), 1.87 - 1.82 (m, 2H), 1.65 (s, 3H), 1.24 (d, J = 8.4 Hz, 1H), 1.21 - 1.11 (m, 12H), 0.89 (dd, J = 9.1, 5.9 Hz, 6H). 13C NMR(101MHz,Chloroform-d)δ170.43,163.78,155.70,149.84,136.67,132.15,130.92,130.73,130.47,12 9.83,128.69,70.25,61.44,54.41,42.88,39.28,37.29,30.84,29.78,24.57,19.25,14.41,13.10,11.86. 31 1H NMR (162MHz, Chloroform-d) δ 145.88. The proton NMR spectrum of compound 10 is shown below. Figure 15 As shown, the carbon spectrum is as follows Figure 16 As shown, the phosphorus spectrum is as follows: Figure 17 As shown.

[0101] It should be noted that in the process of synthesizing compounds 4, 8 and 10, when purifying the products using a silica gel chromatography column, 1% triethylamine needs to be added to the eluent. If 1% triethylamine is not added to the eluent, the product yield will decrease by 20%-30%. Alternatively, if 1% triethylamine is replaced with other substances, such as 1% ammonia, the product yield will also decrease.

[0102] 3) Solid-phase synthesis of protein hydrolysis-targeting chimera A, the synthetic route reaction formula is shown below:

[0103]

[0104] The specific steps are as follows: Under nitrogen protection, compounds 4 and 8 were dissolved in anhydrous dichloromethane to prepare a 0.1M solution, which was then placed in a modification bottle of a German K&A H-8 DNA synthesizer. The sequence was input into the instrument, and a universal solid-phase support (CPG) was used to fill the column at the 3' end of the sequence. After synthesis, the terminal protecting group was removed to obtain A. 1 mL of concentrated ammonia solution was added, and the mixture was heated at 55°C for 8 hours to cleave A from the CPG. After deprotection, the concentrated ammonia solution was removed using a rotary evaporator, and the solution was dissolved in 50% ACN and H2O. The solution was filtered through a 0.22 μm filter and purified by HPLC using a C18 column. After freeze-drying, the solution was reconstituted with dimethyl sulfoxide (DMSO), and the concentration at 260 nm was determined using a Nano Drop microspectrophotometer. The molar amount was calculated, and the molecular weight of the proteolytic targeting chimeric A was found to be 1078.68 Da.

[0105] 4) Solid-phase synthesis of protein hydrolysis targeting chimera B, the synthetic route reaction formula is shown below:

[0106]

[0107] Specific steps are: under the protection of nitrogen, compound 4 and compound 8 are dissolved in anhydrous dichloromethane to form a 0.1M solution, which is placed in a modified bottle of a German K&A H-8 DNA synthesizer, and a thio reagent is prepared into a 0.2M solution and placed in a modified bottle of the thio reagent. The sequence is input on the instrument, a universal CPG is used for column filling at the 3' end of the sequence, after the synthesis is completed, the end protection group is removed to obtain B. 1 mL of concentrated ammonia solution is added, and heating is carried out at 55°C for 8 hours, and B is cut off from the CPG. After deprotection, the concentrated ammonia solution is removed by a rotary evaporator, 50% ACN and H2O are added for dissolution, filtration is performed by using a 0.22 μm filter membrane, HPLC purification is performed by using a C18 chromatographic column. After freeze-drying, DMSO is used for re-dissolution, the concentration at 260 nm is determined by using a microspectrophotometer Nano Drop, the molar mass is calculated, and the molecular weight of the proteolysis targeting chimera B is 1094.68 Da.

[0108] 5) Solid phase synthesis of proteolysis targeting chimera C, the synthesis route reaction formula is as follows:

[0109]

[0110] Specific steps are: under the protection of nitrogen, compound 4 and compound 10 are dissolved in anhydrous dichloromethane to form a 0.1M solution, which is placed in a modified bottle of a German K&A H-8 DNA synthesizer. The sequence is input on the instrument, a universal CPG is used for column filling at the 3' end of the sequence, after the synthesis is completed, the end protection group is removed to obtain C. 1 mL of 50 mM potassium carbonate methanol solution is added, and heating is carried out at room temperature for 8 hours, and C is cut off from the CPG. After deprotection, 6 μL of acetic acid is added to the deprotection solution, mixed, the potassium carbonate methanol solution is removed by a rotary evaporator, 50% ACN and H2O are added for dissolution, filtration is performed by using a 0.22 μm filter membrane, HPLC purification is performed by using a C18 chromatographic column. After freeze-drying, DMSO is used for re-dissolution, the concentration at 260 nm is determined by using a microspectrophotometer Nano Drop, the molar mass is calculated, and the molecular weight of the proteolysis targeting chimera D is 1134 Da.

[0111] 6) Solid phase synthesis of proteolysis targeting chimera D, the synthesis route reaction formula is as follows:

[0112]

[0113] The specific steps are: under the protection of nitrogen, compound 4 and compound 10 are dissolved in anhydrous dichloromethane to prepare a 0.1M solution in a modified bottle of German K&AH-8 DNA synthesizer. Input the sequence on the instrument, use 3' phosphate CPG solid phase carrier at the 3' end of the sequence for column filling, and after synthesis, remove the end protection group to obtain D. Add 1 mL of 50 mM potassium carbonate methanol solution, and cut D and C from CPG after 8 hours at room temperature. After deprotection, 6 μL of acetic acid is added to the deprotection solution, mixed, and then the potassium carbonate methanol solution is removed by rotary evaporation, dissolved with ultrapure water, filtered with a 0.22 μm filter membrane, and purified by HPLC using a C18 column. After freeze-drying, resuspend in 1% DMSO, measure the concentration at 260 nm using a microspectrophotometer Nano Drop, and calculate the molar mass to obtain the molecular weight of the proteolysis targeting chimera D as 1214.3 Da.

[0114] It should be noted that in the above process of synthesizing proteolysis targeting chimeras A, B, C, and D by DNA synthesizer, changing the coupling times, coupling time, and modified monomer concentration will affect the yield of the synthesized proteolysis targeting chimeras.

[0115] Example 2, Effect of Proteolysis Targeting Chimeras Containing Different Linkers on Inhibiting HeLa Cells

[0116] The proteolysis targeting chimeras A, B, and C prepared in Example 1 differ in their linkers, which are phosphodiester, phosphorothioate, and phosphotriester, respectively. This example explores the effects of these three proteolysis targeting chimeras containing different linkers and MZ1 on inhibiting human cervical cancer HeLa cells. The MZ1 is a commonly used proteolysis targeting chimera targeting BRD4, and its linker is polyethylene glycol, with the following structure:

[0117]

[0118] The experimental steps are as follows: HeLa cells are seeded in a 96-well plate at a density of 5*10 3Cells were cultured in wells at 37°C and 5% CO2 for 12 hours. Then, culture medium containing 0 nM, 10 nM, 50 nM, 200 nM, 500 nM, 1000 nM, and 2000 nM proteolytic targeting chimeras (A, B, C, and MZ1) was added, and the cells were cultured at 37°C and 5% CO2 for 48 hours. After incubation, the drug-containing medium was removed, and the cells were incubated in medium containing 10% CCK-8 for 1 hour to determine the inhibitory effect. The inhibitory effect was analyzed using the CCK-8 (Cell Counting kit-8) assay. The absorbance at 450 nm was measured using a FlexStation microplate reader, and cell viability (V) was calculated using the following formula:

[0119] V = [(RA-RE) / (RB-RE)] × 100%

[0120] Note: In the formula, RA, RB, and RE represent the absorbance of the experimental group, the blank group, and the solvent-based color group, respectively. Results are as follows: Figure 18 As shown.

[0121] Depend on Figure 18 It can be seen that the IC50 of the protein hydrolysis-targeting chimeric MZ1 is... 50 The value was 385.9 nM, and the IC50 of the protein hydrolysis targeting chimeric C was... 50 The value was 242.9 nM. The inhibitory effect of the proteolytic targeted chimera C on human cervical cancer HeLa cells was better than that of the proteolytic targeted chimera MZ1. However, the proteolytic targeted chimeras A and B had almost no inhibitory effect on human cervical cancer HeLa cells. It is speculated that this is because the phosphodiester backbone of the proteolytic targeted chimera A and the thiophosphate backbone of the proteolytic targeted chimera B are both negatively charged, resulting in poor cell permeability and difficulty in entering the cells to exert their effects. On the other hand, the phosphotriester backbone of the proteolytic targeted chimera C and the polyethylene glycol backbone of the proteolytic targeted chimera MZ1 are both electrically neutral, with good cell permeability, and can enter human cervical cancer HeLa cells, thus inhibiting cell proliferation of human cervical cancer HeLa cells.

[0122] Depend on Figure 19 It can be seen from the HPLC chromatogram comparison of the protein hydrolysis-targeting chimera MZ1 and C that the protein hydrolysis-targeting chimera C is more hydrophobic than MZ1, making it easier to penetrate the cell membrane. Therefore, the IC50 of the protein hydrolysis-targeting chimera C is higher. 50 The IC50 value of the protein hydrolysis-targeting chimeric MZ1 was compared to that of the other chimeric protein. 50 The value is lower, so it has a stronger inhibitory effect on human cervical cancer HeLa cells than the protein hydrolysis-targeting chimeric C.

[0123] The above experiment proves that the proteolysis targeting chimera C containing the electrically neutral phosphotriester skeleton has a stronger inhibitory effect on human cervical cancer HeLa cells. The modification group of the phosphotriester in the proteolysis targeting chimera C is isobutyl. Further, the modification group of the phosphotriester in the proteolysis targeting chimera C is changed, and the modification group includes methyl, ethyl, propyl, isopropyl, and the like. The effect of the proteolysis targeting chimera containing different neutral linkers on inhibiting human cervical cancer HeLa cells is detected according to the above experimental method. The experimental results prove that the proteolysis targeting chimeras prepared by containing the above other modification groups have an effect on inhibiting human cervical cancer HeLa cells, which is not as good as that of the proteolysis targeting chimera C.

[0124] Preferably, the linker for preparing the proteolysis targeting chimera is an isobutyl phosphotriester.

[0125] Example 3, Effect of proteolysis targeting chimera C and MZ1 on degrading BRD4 protein in HeLa cells

[0126] The experiment in Example 2 proves that the proteolysis targeting chimera C and MZ1 both have a good cell proliferation inhibitory effect on human cervical cancer HeLa cells, and the proteolysis targeting chimera C has a better inhibitory effect than the other. The following experiment will detect the effect of the proteolysis targeting chimera C and MZ1 on degrading BRD4 protein in human cervical cancer HeLa cells through Western Blotting experiment.

[0127] The specific experimental steps are as follows:

[0128] HeLa cells were inoculated in a 12-well plate at 1*10 5 After 12 hours of culture at 37°C in a 5% carbon dioxide condition, the cells were treated with proteolysis targeting chimera C and MZ1 at concentrations of 3.16 nM, 10 nM, 31.6 nM, 100 nM, 316 nM, and 1000 nM, respectively. After 24 hours of drug treatment, the culture medium was aspirated, and the cells were washed twice with PBS. Then, 90 μl of RIPA lysis solution was added, and the protein was extracted on ice. The protein concentration was measured by BCA protein quantification method, and the bromophenol blue was boiled after being added. The amount of protein loaded on each well was 20 μl. The electrophoresis was stopped when the bromophenol blue dye was electrophoresed to the bottom of the gel. Wet transfer was used. After transfer, the membrane was washed with TBST for 3 times, 10 min each time, and blocked with 5% skimmed milk powder for 2 hours. The BRD4 (1:1000) and GAPDH (1:1000) primary antibodies were added and incubated at 4°C overnight. The next day, the membrane was washed with TBST for 3 times, 10 min each time, and the corresponding secondary antibody (1:10000) was added and incubated at room temperature for 1 hour. The membrane was washed with TBST for 3 times, 10 min each time. ECL developing solution was used for color development. The results of the proteolysis targeting chimera C and MZ1 degrading BRD4 protein are as follows: Figure 20As shown, the degradation curve of BRD4 protein in human cervical cancer HeLa cells by the proteolytic targeting chimera MZ1 is as follows: Figure 21 As shown, the degradation curve of BRD4 protein in human cervical cancer HeLa cells by the proteolytic targeting chimera C is as follows: Figure 22 As shown.

[0129] Depend on Figure 20 It can be seen that, after treatment of cells with different concentrations of proteolytic targeting chimera C and MZ1, the bands of residual BRD4 in the cells were darker than those of the corresponding control group treated with 1% DMSO. This indicates that both proteolytic targeting chimera C and MZ1 can effectively degrade BRD4 protein in cells.

[0130] Depend on Figure 21 and Figure 22 It can be seen that the DC of protein hydrolysis targets chimeric C. 50 The value is 80.16 nM, the DC value of the protein hydrolysis targeting chimeric MZ1. 50 The value was 104.4 nM, which, compared with the proteolytic targeting chimera MZ1, indicates that the proteolytic targeting chimera C has a better degradation effect on BRD4 protein in human cervical cancer HeLa cells, consistent with the results of Example 2.

[0131] Example 4: Detection of the dephosphorylation performance of protein hydrolysis-targeting chimera D

[0132] In Example 1, proteolytic targeting chimera C and proteolytic targeting chimera D were prepared. The difference between them is that proteolytic targeting chimera D, after dephosphorylation by alkaline phosphatase, forms proteolytic targeting chimera C. The reaction formula is as follows:

[0133]

[0134] 1. To verify whether dephosphorylation of protein hydrolysis-targeting chimera D yields protein hydrolysis-targeting chimera C, protein hydrolysis-targeting chimera D, protein hydrolysis-targeting chimera D treated with alkaline phosphatase, and protein hydrolysis-targeting chimera C were compared by mass spectrometry and HPLC. The protein hydrolysis-targeting chimera D and protein hydrolysis-targeting chimera C used in the following experiments were prepared according to the method described in Example 1.

[0135] The experimental method is as follows: the proteolysis targeting chimera D is diluted with a buffer solution (100 mM Tris-HCl (pH=8.0 at 37℃), 50 mM MgCl2, 1 M KCl, 0.2% Triton X-100, 10 mM 2-mercaptoethanol, 1 mg / mL BSA) to a final concentration of 20 μM, and then 0.5 U / mL ALP is added to the above solution. After mixing, the solution is incubated at 37℃ for 10 minutes, and then at 75℃ for 5 minutes to inactivate the ALP. Filtration is performed with a 0.22 μm filter, and analytical purification is performed on an LC20 AR Shimadzu HPLC using a C18 chromatographic column.

[0136] The mass spectrum of the proteolysis targeting chimera C is shown in Figure 23 The mass spectrum of the proteolysis targeting chimera D is shown in Figure 24 The mass spectrum of the proteolysis targeting chimera D after alkaline phosphatase treatment is shown in Figure 25 The liquid chromatogram obtained after HPLC treatment of the three substances is shown in Figure 26

[0137] As can be seen from the comparison of the mass spectra of Figure 23 , 24 , 25, the mass spectrum of the proteolysis targeting chimera D after alkaline phosphatase treatment ( Figure 25 ) is consistent with the mass spectrum of the proteolysis targeting chimera C ( Figure 23 ), indicating that the proteolysis targeting chimera D dephosphorylates to form the proteolysis targeting chimera C after co-incubation with alkaline phosphatase. As can be seen from Figure 26 , the peak time of the proteolysis targeting chimera D is 28.5 min, the peak time of the proteolysis targeting chimera C is 32.5 min, and the peak time of the proteolysis targeting chimera D after alkaline phosphatase treatment is 32.5 min, indicating that the proteolysis targeting chimera D dephosphorylates to form the proteolysis targeting chimera C after co-incubation with alkaline phosphatase.

[0138] ​2. To investigate the effect of different concentrations of alkaline phosphatase on the dephosphorylation of proteolytic targeted chimera D to form proteolytic targeted chimera C, the experimental method was as follows: Proteolytic targeted chimera D was diluted to a final concentration of 20 μM with a buffer solution (100 mM Tris-HCl (pH = 8.0 at 37℃), 50 mM MgCl2, 1 M KCl, 0.2% Triton X-100, 10 mM 2-mercaptoethanol, 1 mg / mL BSA). Then, 2.5 U / mL, 0.5 U / mL, and 0.05 U / mL of ALP were added to the proteolytic targeted chimera D solution, respectively, with the solution of proteolytic targeted chimera D without ALP treatment serving as a control. After mixing, the solution was incubated at 37℃ for 10 minutes, and then at 75℃ for 5 minutes to inactivate ALP. The solution was filtered through a 0.22 μm filter membrane and purified by analysis using a C18 column on an LC20AR Shimadzu HPLC system. The liquid phase spectrum obtained after HPLC processing is shown below. Figure 27 As shown, by Figure 27 It can be seen that as the concentration of alkaline phosphatase increases, the proportion of proteolytic targeted chimera C obtained by dephosphorylation of proteolytic targeted chimera D is higher. When the concentration of alkaline phosphatase is 0.5 U / mL, almost all of the proteolytic targeted chimera D is dephosphorylated to obtain proteolytic targeted chimera C, indicating that proteolytic targeted chimera C will be generated by dephosphorylation with alkaline phosphatase.

[0139] Compared to normal cells, tumor and other diseased tissue cells have high ALP expression on their cell membrane surface. Therefore, the protein hydrolysis targeting chimera D can exert its function after being dephosphorylated by ALP on the cell membrane surface of these diseased tissue cells.

[0140] 3. To verify whether the alkaline phosphatase inhibitor Na3VO4 can inhibit the dephosphorylation of proteolytic targeted chimera D to obtain proteolytic targeted chimera C, the specific experimental steps were as follows: Proteolytic targeted chimera D was diluted to a final concentration of 20 μM with a buffer solution (100 mM Tris-HCl (pH = 8.0 at 37℃), 50 mM MgCl2, 1 M KCl, 0.2% Triton X-100, 10 mM 2-mercaptoethanol, 1 mg / mL BSA). Then, 0.5 U / mL ALP + (2 mM) Na3VO4 and 0.5 U / mL ALP were added to the proteolytic targeted chimera D solution, respectively, with the solution of proteolytic targeted chimera D without any additives as a control. After mixing, the solution was incubated at 37℃ for 10 minutes, and then at 75℃ for 5 minutes to inactivate ALP. The sample was filtered through a 0.22 μm filter membrane and analyzed and purified using a C18 column on an LC20AR Shimadzu HPLC system. The resulting liquid chromatography chromatogram is shown below.Figure 28 Figure 1 shows the HPLC chromatogram of the proteolysis targeting chimera D.

[0141] Figure 2 shows the HPLC chromatogram of the proteolysis targeting chimera C. Figure 28 As can be seen, the peak time of the proteolysis targeting chimera D with the addition of ALP and Na3VO4 was 28.5 min, which was the same as the peak time of the proteolysis targeting chimera D in the control group. The peak time of the proteolysis targeting chimera D with the addition of ALP was 32.5 min, indicating that ALP had converted the proteolysis targeting chimera D into the proteolysis targeting chimera C. The above experimental results show that the alkaline phosphatase inhibitor Na3VO4 can effectively inhibit the dephosphorylation of the proteolysis targeting chimera D, and at the same time verify that the conversion of the proteolysis targeting chimera D into the proteolysis targeting chimera C is due to the dephosphorylation of the proteolysis targeting chimera D by alkaline phosphatase.

[0142] Example 5, detection of the stability of the proteolysis targeting chimera C formed after the dephosphorylation of the proteolysis targeting chimera D in the plasma

[0143] In order to verify the stability of the proteolysis targeting chimera C obtained by dephosphorylation of the proteolysis targeting chimera D in the plasma, the proteolysis targeting chimera C (20 μM) and MZ1 (20 μM) prepared in Example 1 were incubated in 1640 medium added with 10% fetal bovine serum at 37°C, and the control group was 1640 medium added with 10% fetal bovine serum. The incubation time was 0, 6, 12, 24 and 48 hours. After the incubation was completed, acetonitrile was used for dilution, 0.22 μm filter membrane was used for filtration, and HPLC analysis was performed on LC20AR Shimadzu using a C18 chromatographic column.

[0144] The liquid chromatogram of the extracted proteolysis targeting chimera C was as shown in Figure 3 and Table 2 below, and the stability curve of the proteolysis targeting chimera C was as shown in Figure 4. Figure 29 Figure 30 The liquid chromatogram of the extracted MZ1 was as shown in Figure 5 and Table 3 below, and the stability curve of MZ1 was as shown in Figure 6. Figure 31 Figure 32

[0145] Table 2, liquid phase analysis results of the stability of the proteolysis targeting chimera C in the medium containing 10% fetal bovine serum

[0146] Experimental group Target peak area Proportion PROTAC C-0h 3456225 100％ PROTAC C-6h 3781756 109.42％ PROTAC C-12h 2550000 73.78％ PROTAC C-24h 2314369 66.96％ PROTAC C-48h 2351451 68.04％

[0147] Table 3, liquid phase analysis results of the stability of MZ1 in the medium containing 10% fetal bovine serum

[0148] Experimental group Target peak area Proportion MZ1-0h 5735074 100％ MZ1-6h 5536363 96.54％ MZ1-12h 6318953 110.18％ MZ1-24h 5213027 90.90％ MZ1-48h 4909312 85.60％

[0149] According to the above Tables 2-3 and Figure 29-32 ​​​Analysis, within the first 24 hours, proteolysis targeting chimera C and MZ1 present different degrees of degradation, but proteolysis targeting chimera C remains stable at about 65% after 24 hours, while MZ1 has been declining after 24 hours, indicating that proteolysis targeting chimera C has good stability in serum-containing medium.

[0150] Example 6, Effect of proteolysis targeting chimera D on tumor cells expressing different contents of alkaline phosphatase

[0151] This example investigates the effect of proteolysis targeting chimera D on high alkaline phosphatase-expressing tumor cells and low alkaline phosphatase-expressing tumor cells, respectively. The high alkaline phosphatase-expressing tumor cells are human osteosarcoma Saos-2 cells, and the low alkaline phosphatase-expressing tumor cells are human osteosarcoma U-2OS cells.

[0152] 1. Determination of alkaline phosphatase activity of human osteosarcoma Saos-2 cells and human osteosarcoma U-2OS cells

[0153] The determination method is as follows: Saos-2 cells and U-2OS cells are inoculated in 6-well plates, 3*10 5 cells / well, and cultured at 37°C in a 5% carbon dioxide environment for 24 hours. Then the cells are washed with Tris-HCl buffer (pH = 7.4). Add 200 μl RIPA lysis buffer (without enzyme inhibitor), lyse the cells on ice to extract proteins, and determine the protein concentration by BCA protein quantification method. Use the Biyun Tian alkaline phosphatase detection kit, set up blank control wells, standard wells and sample wells, and detect 10 μg of protein. After incubation at 37°C for 30 minutes, add the reaction termination solution, and measure the absorbance at 405 nm to obtain the results as shown in Figure 33 .

[0154] As can be seen from Figure 33 , under the condition of 37°C, the production of p-nitrophenol per milligram of protein per minute in Saos-2 cells and U-2OS cells is 3.217 μmol and 0.117 μmol, respectively, indicating that alkaline phosphatase is highly expressed in Saos-2 cells and lowly expressed in U-2OS cells, and the alkaline phosphatase content of Saos-2 cells is more than 25 times higher than that of U-2OS cells.

[0155] 2. Inhibitory effect of proteolysis targeting chimera D on Saos-2 cells and U-2OS cells

[0156] The protein hydrolysis-targeting chimera D prepared in Example 1 was added as an additive to Saos-2 cells and U-2OS cells respectively to detect its cell inhibition effect, and the protein hydrolysis-targeting chimera C prepared in Example 1 without enzyme activator group modification was used as a comparison.

[0157] The experimental procedure for detecting the inhibitory effects of proteolytic-targeting chimera D and proteolytic-targeting chimera C on Saos-2 and U-2OS cells was as follows: Saos-2 cells and U-2OS cells were seeded into 96-well plates, 5*103 3 / well, cultured at 37℃, 5% CO2 for 12 hours. Proteolytic targeting chimera D and proteolytic targeting chimera C at concentrations of 0 nM, 200 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM, and 8000 nM were added to the above two cell types, respectively, and cultured at 37℃, 5% CO2 for 48 hours. After incubation, the drug-containing medium was removed, and the cells were incubated with medium containing 10% CCK-8 for 1 hour to determine the inhibitory effect of the drug. The inhibitory effect was analyzed using the CCK-8 (Cell Counting kit-8) assay, with absorbance measured at 450 nm using a FlexStation microplate reader. Cell viability (V) was calculated using the following formula:

[0158] V = [(RA-RE) / (RB-RE)] × 100%

[0159] Note: In the formula, RA, RB, and RE represent the absorbance of the experimental group, the blank group, and the solvent-based color group, respectively.

[0160] The inhibitory effects of proteolytic targeting chimera D and proteolytic targeting chimera C on Saos-2 cells are as follows: Figure 34 As shown, the inhibitory effect on U-2OS cells is as follows: Figure 35 As shown.

[0161] Depend on Figure 34-35 It can be seen that in human osteosarcoma Saos-2 cells with high alkaline phosphatase expression, the IC50 of proteolytic targeting chimeric D is [not specified]. 50 The value was 1364 nM, and the IC50 of the proteolytic targeting chimeric C was... 50 The value was 931.2 nM, indicating that proteolytic targeting chimeras D and C had a good inhibitory effect on the proliferation of human osteosarcoma Saos-2 cells; however, in human osteosarcoma U-2OS cells with low alkaline phosphatase expression, proteolytic targeting chimera D failed to show an IC50 value. 50 Value, IC50 of protein hydrolysis targeting chimeric C 50The value is 3128 nM, indicating that only proteolysis targeting chimera C has inhibitory effect on human osteosarcoma U-2OS cells, and proteolysis targeting chimera D does not. The above experimental results show that proteolysis targeting chimera D is selective for cells and can only inhibit the growth of Saos-2 cells with high expression of alkaline phosphatase.

[0162] Proteolysis targeting chimera D can only play a role in dephosphorylation to form proteolysis targeting chimera C in human osteosarcoma Saos-2 cells with high expression of alkaline phosphatase to degrade BRD4 protein to inhibit cell proliferation; in human osteosarcoma U-2OS cells, the expression content of alkaline phosphatase is very low, and the amount of proteolysis targeting chimera D dephosphorylated to form proteolysis targeting chimera C is very small and can be ignored, so proteolysis targeting chimera D cannot play a role in osteosarcoma U-2OS cells. Proteolysis targeting chimera C does not have a modified enzyme activatable group and can directly play a role in degrading BRD4 protein to inhibit cell proliferation, so proteolysis targeting chimera C has no selectivity for cells and can inhibit both Saos-2 cells with high expression of alkaline phosphatase and U-2OS cells with low expression of alkaline phosphatase.

[0163] 3. Effect of proteolysis targeting chimera D on degradation of BRD4 protein in Saos-2 cells and U-2OS cells

[0164] The above 2 proves that proteolysis targeting chimera D is selective for cells and can only dephosphorylate and further degrade BRD4 protein to inhibit cell proliferation in Saos-2 cells with high expression of alkaline phosphatase. This experiment further detects the effect of proteolysis targeting chimera D on degradation of BRD4 protein in Saos-2 cells and U-2OS cells by Western Blotting experiment, with proteolysis targeting chimera C as a comparison.

[0165] The specific experimental steps are as follows: Saos-2 cells and U-2OS cells were inoculated in 12-well plates, 1*10 5 / well, after 12 hours of incubation at 37°C in 5% carbon dioxide, the two kinds of cells were treated with proteolysis targeting chimera D and proteolysis targeting chimera C at concentrations of 3.16 nM, 10 nM, 31.6 nM, 100 nM, 316 nM and 1000 nM, respectively. After 24 hours of drug treatment, the culture medium was removed, and the cells were washed twice with PBS. Then, 90 μl of RIPA lysis solution was added, and the protein was extracted on ice. The protein concentration was determined by BCA protein quantification method, and the bromophenol blue was boiled after being added. The amount of protein loaded on each well was 20 μl. The electrophoresis was stopped when the bromophenol blue dye was electrophoresed to the bottom of the gel. Wet transfer was used, and after transfer, the membrane was washed with TBST for 10 min three times. The membrane was blocked with 5% skim milk for 2 h. BRD4 (1:1000) and GAPDH (1:1000) primary antibodies were added and incubated at 4°C overnight. The next day, the membrane was washed with TBST for 10 min three times, and the corresponding secondary antibody (1:10000) was added and incubated at room temperature for 1 h. The membrane was washed with TBST for 10 min three times. ECL developing solution was used for color development. The Western Blotting results of BRD4 protein degraded by proteolysis targeting chimera D and proteolysis targeting chimera C in Saos-2 cells are shown in Figure 36 , and the degradation curve is shown in Figure 37-38 . The Western Blotting results of BRD4 protein degraded by proteolysis targeting chimera D and proteolysis targeting chimera C in U-2 OS cells are shown in Figure 39 , and the degradation curve is shown in Figure 40-41 .

[0166] As can be seen from Figure 36-38 , the Western Blotting results show that there is no significant difference in the remaining BRD4 protein bands in the two groups of human osteosarcoma Saos-2 cells treated with proteolysis targeting chimera D and proteolysis targeting chimera C at concentrations of 3.16 nM, 10 nM, 31.6 nM, 100 nM, 316 nM and 1000 nM. With the increase of the concentration of proteolysis targeting chimera, the remaining BRD4 protein content decreases. The DC 50 value of proteolysis targeting chimera D is 886.7 nM, and the DC 50 value of proteolysis targeting chimera C is 303 nM, indicating that proteolysis targeting chimera D and proteolysis targeting chimera C have good degradation effect on BRD4 protein in human osteosarcoma Saos-2 cells.

[0167] As can be seen from Figure 39-41It can be seen that the Western Blotting results show that there is a significant difference in the remaining BRD4 protein bands in the two groups of human osteosarcoma U-2OS cells after being treated with different concentrations of proteolysis targeting chimera D and proteolysis targeting chimera C. The remaining BRD4 protein in the human osteosarcoma U-2OS cells treated with different concentrations of proteolysis targeting chimera D decreases little with the increase of the concentration of proteolysis targeting chimera D, which can be almost ignored, and proteolysis targeting chimera D fails to detect DC 50 values, indicating that proteolysis targeting chimera D has no obvious degradation effect on BRD4 protein in human osteosarcoma U-2OS cells. The remaining BRD4 protein in the human osteosarcoma U-2OS cells treated with different concentrations of proteolysis targeting chimera C significantly decreases with the increase of the concentration of proteolysis targeting chimera C, and the DC 50 values of proteolysis targeting chimera C is 261.7 nM, indicating that proteolysis targeting chimera C has an obvious degradation effect on BRD4 protein in human osteosarcoma U-2OS cells.

[0168] Through the above experiments and analysis, it is proved that proteolysis targeting chimera D in the two kinds of human osteosarcoma Saos-2 cells and human osteosarcoma U-2OS cells with significant difference in alkaline phosphatase expression, the dephosphorylation in the alkaline phosphatase high expression Saos-2 cells is selected to realize the efficient degradation of BRD4 protein, and the anti-tumor effect is achieved, which shows that the proteolysis targeting chimera provided by the present application can effectively prevent the E3 ubiquitin ligase ligand from recruiting E3 ubiquitin ligase to degrade the protein by modifying an enzyme activatable group on the hydroxyl group of the E3 ubiquitin ligase ligand. Only when the corresponding enzyme of the enzyme activatable group removes the enzyme activatable group, the proteolysis targeting chimera can play its role, so as to realize the degradation of the target protein after the proteolysis targeting chimera selects tumor cells.

[0169] Although the present application is disclosed as above, the present application is not limited thereto. Any person skilled in the art can make various modifications and changes without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be subject to the scope defined by the claims.

Claims

1. A method of making a proteolysis targeting chimera, characterized by, Prepared by DNA automated solid-phase synthesis method, comprising the following steps: (1) preparing and purifying E3 ubiquitin ligase ligand monomer containing phosphoramidite structure; (2) preparing and purifying target protein ligand monomer containing phosphoramidite structure; (3) synthesizing E3 ubiquitin ligase ligand monomer containing phosphoramidite structure and target protein ligand monomer containing phosphoramidite structure on a solid-phase carrier by a DNA synthesizer, and after deprotection and purification treatment, obtaining a proteolysis targeting chimera; The structural formula of the proteolysis targeting chimera is as follows:

2. The production method according to claim 1, wherein The purification method in steps (1) and (2) is both by chromatography column purification, and the eluent of the chromatography column comprises triethylamine.

3. The production method according to claim 2, wherein The solid-phase carrier in step (3) comprises a general solid-phase carrier or a solid-phase carrier containing an enzyme-activatable group, and the enzyme-activatable group comprises one or more of a phosphate group, a nitro group.

4. Use of a proteolysis targeting chimera for the manufacture of a medicament for the treatment of cervical cancer and osteosarcoma, characterized in that, The proteolytic targeting chimera has the structure 5. Use of a proteolysis targeting chimera for the manufacture of a medicament for the treatment of human osteosarcoma, characterized in that, The proteolytic targeting chimera has the structure

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